Methods for treating cancers and pathogen infections using antigen-presenting cells loaded with RNA

ABSTRACT

Disclosed are cells and methods for treating or preventing tumor formation or infections with pathogens in a patient. The cells of the invention are antigen-presenting cells (e.g., dendritic cells or macrophage) that have been loaded with RNA derived from tumors or pathogens. By administering the RNA-loaded antigen-presenting cells to a patient, tumor formation or pathogen infections can be treated or prevented. Alternatively, the RNA-loaded cells can be used as stimulator cells in the ex vivo expansion of CTL. Such CTL can then be used in a variation of conventional adoptive immunotherapy techniques.

This application is a continuation of application Ser. No. 08/640,444,filed Apr. 30, 1996, now U.S. Pat. No. 5,853,719.

BACKGROUND OF THE INVENTION

This invention relates to methods for treating or preventing tumorformation or pathogen infection in a patient.

Previously-described methods for treating cancers include the use ofchemotherapeutics, radiation therapy, and selective surgery. Theidentification of a few tumor antigens has led to the development ofcell-based therapies. These methods rely on first identifying a tumorantigen (i.e., a polypeptide that is expressed preferentially in tumorcells, relative to non-tumor cells). Several human tumor antigens havebeen isolated from melanoma patients, and identified and characterized(Boon and van der Bruggen, 1996, J. Exp. Med. 183: 725-729). Thesepolypeptide antigens can be loaded onto antigen-presenting cells, andthen be administered to patients in a method of immunotherapy (i.e., asa vaccine) Alternatively, the polypeptide-loaded antigen-presentingcells can be used to stimulate CTL proliferation ex vivo. The stimulatedCTL are then administered to the patient in a method of adoptiveimmunotherapy.

A variety of methods have been described for treating infections withintracellular pathogens such as viruses and bacteria. For example,antibiotics are commonly used to treat bacterial infections.Preparations of killed pathogens can also serve as vaccines. Inaddition, CTL-based therapies have been described for treating suchinfections.

SUMMARY OF THE INVENTION

Applicants have discovered that tumor formation in a patient can betreated or prevented by administering to the patient anantigen-presenting cell(s) that is loaded with antigen encoded in RNAderived from a tumor. For convenience, an RNA-enriched tumor preparationcan be used in lieu of purified RNA. The invention thus circumvents theneed purify RNA or isolate and identify a tumor antigen. Using similarmethods and pathogen-derived RNA, pathogen infection in a patient can betreated or prevented. The RNA-loaded antigen-presenting cells can beused to stimulate CTL proliferation ex vivo or in vivo. The ex vivoexpanded CTL can be administered to a patient in a method of adoptiveimmunotherapy.

Accordingly, the invention features a method for producing an RNA-loadedantigen-presenting cell (APC); the method involves introducing into anAPC in vitro (i) tumor-derived RNA that includes tumor-specific RNA or(ii) pathogen-derived RNA that includes pathogen-specific RNA (e.g.tumor-specific RNA of an intracellular pathogen), thereby producing anRNA-loaded APC. Upon introducing RNA into an APC (i.e., “loading” theAPC with RNA), the RNA is translated within the APC, and the resultingprotein is processed by the class I processing and presentation pathway.Presentation of RNA-encoded peptides begins the chain of events in whichthe immune system mounts a response to the presented peptides.

Preferably, the APC is a professional APC such as a dendritic cell or amacrophage. Alternatively, any APC can be used. For example, endothelialcells and artificially generated APC can be used. The RNA that is loadedonto the APC can be provided to the APC as purified RNA, or as afractionated preparation of a tumor or pathogen. The RNA can includepoly A⁺ RNA, which can be isolated by using conventional methods (e.g.,use of poly dT chromatography). Both cytoplasmic and nuclear RNA isuseful in the invention.. Also useful in the invention is RNAcorresponding to defined tumor or pathogen antigens or epitopes, and RNAcorresponding to “minigenes” (i.e., RNA sequences encoding definedepitopes). If desired, tumor-specific or pathogen-specific RNA can beused; such RNA can be prepared using art-known techniques such assubtractive hybridization against RNA from non-tumor cells or againstrelated, but non-pathogenic, bacteria or viruses.

The RNA that is loaded onto APC can be isolated from a cell, or it canbe produced by employing conventional molecular biology techniques. Forexample, RNA can be extracted from tumor cells, reverse transcribed intocDNA, which can be amplified by PCR, and the cDNA then is transcribedinto RNA to be used in the invention. If desired, the cDNA can be clonedinto a plasmid before it is used as a template for RNA synthesis. Suchtechniques allow one to obtain large amounts of the RNA antigen from asmall number of cells, which is particularly advantageous because tumorpatients often have few tumor cells. In one embodiment, the APC arecontacted with the tumor-derived RNA in the presence of a cationiclipid, such as DOTAP or 1:1 (w/w) DOTMA:DOPE (i.e., LIPOFECTIN).Alternatively, art-known transfection methods are used to introduce theRNA into the APC.

Because practicing the invention does not require identifying an antigenof the tumor cell or pathogen, RNA derived from essentially any type oftumor or pathogen is useful. For example, the invention is applicable,but not limited, to the development of therapeutics for treatingmelanomas, bladder cancers, breast cancers, pancreatic cancers, prostatecancers, colon cancers, and ovarian cancers. In addition, the inventioncan treat or prevent infections with pathogens such as Salmonella,Shigella, Enterobacter, human immunodeficiency virus, Herpes virus,influenza virus, poliomyelitis virus, measles virus, mumps virus, orrubella virus.

The antigen-presenting cells produced in accordance with the inventioncan be used to induce CTL responses in vivo and ex vivo. Thus, theinvention includes methods for treating or preventing tumor formation ina patient by administering to the patient a therapeutically effectiveamount of APC loaded with tumor-derived RNA. The tumor-derived RNA canbe derived from the patient, e.g., as an RNA-enriched tumor preparation.Alternatively, the tumor-derived RNA used in such a treatment regimencan be derived from another patient afflicted with the same, or asimilar, type of cancer. Likewise, APC loaded with pathogen-derived RNAcan be used to treat or prevent a pathogen infection in a patient.

Included within the invention are methods for producing a cytotoxic Tlymphocyte. Such a CTL can be produced by contacting a T lymphocyte invitro with an antigen-presenting cell that is loaded with tumor-derivedor pathogen-derived RNA, and maintaining the T lymphocyte underconditions conducive to CTL proliferation, thereby producing a CTL. Theresulting CTL show remarkable specificity for the pathogen or the cellsof the tumor from which the loaded RNA is derived. Such CTL can beadministered to a patient in a variation of conventional adoptiveimmunotherapy methods.

The invention also includes methods for treating or preventing tumorformation in a patient by administering to the patient a therapeuticallyeffective amount of APC loaded with tumor-derived RNA. Similarly, theinvention provides methods for treating pathogen infection in a patientby administering to the patient a therapeutically effective amount ofAPC loaded with pathogen-derived RNA. The T lymphocytes that are used inthese various therapeutic methods can be derived from the patient to betreated, or haplotype-matched CTL from a donor can be used. Similarly,the RNA used in these methods can be derived from the patient to betreated, or RNA from a donor can be used.

By “RNA-loaded” or “RNA-pulsed” antigen-presenting cell is meant an APC(e.g., a macrophage or dendritic cell) that was incubated or transfectedwith RNA, e.g., RNA derived from a tumor or pathogen. Such RNA can beloaded onto the APC by using conventional nucleic acid transfectionmethods, such as lipid-mediated transfection, electroporation, andcalcium phosphate transfection. For example, RNA can be introduced intoAPC by incubating the APC with the RNA (or extract) for 1 to 24 hours(e.g., 2 hours) at 37° C., preferably in the presence of a cationiclipid.

By “tumor-derived” RNA is meant a sample of RNA that has its origin in atumor cell, and which includes RNA corresponding to a tumor antigen(s).Included is RNA that encodes all or a portion of a previously identifiedtumor antigen. Similarly “pathogen-derived” RNA is a sample of RNA thathas its origin in an pathogen (e.g., a bacterium or virus, includingintracellular pathogens). Such RNA can be “in vitro transcribed,” e.g.,reverse transcribed to produce cDNA that can be amplified by PCR andsubsequently be transcribed in vitro, with or without cloning the cDNA.Also included is RNA that is provided as a fractionated preparation oftumor cell or pathogen. Because even unfractionated RNA preparation(e.g., total RNA or total poly A+ RNA) can be used, it is not necessarythat a tumor or pathogen antigen be identified. In one embodiment, thepreparation is fractionated with respect to a non-RNA component(s) ofthe cell in order to decrease the concentration of a non-RNA component,such as protein, lipid, and/or DNA and enrich the preparation for RNA.If desired, the preparation can be further fractionated with respect tothe RNA (e.g., by subtractive hybridization) such that “tumor-specific”or “pathogen-specific” RNA is produced.

By “tumor-specific” RNA is meant an RNA sample that, relative tounfractionated tumor-derived RNA, has a high content of RNA that ispreferentially present in a tumor cell compared with a non-tumor cell.For example, tumor-specific RNA includes RNA that is present in a tumorcell, but not present in a non-tumor cell. Also encompassed in thisdefinition is an RNA sample that includes RNA that is present both intumor and non-tumor cells, but is present at a higher level in tumorcells than in non-tumor cells. Also included within this definition isRNA that encodes a previously identified tumor antigen and which isproduced in vitro, e.g., from a plasmid or by PCR. Alternatively,tumor-specific RNA can be prepared by fractionating an RNA sample suchthat the percentage of RNA corresponding to a tumor antigen isincreased, relative to unfractionated tumor-derived RNA. For example,tumor-specific RNA can be prepared by fractionating tumor-derived RNAusing conventional subtractive hybridization techniques against RNA fromnon-tumor cells. Likewise, “pathogen-specific” RNA refers to an RNAsample that, relative to unfractionated pathogen-derived RNA, has a highcontent of RNA that is preferentially present in the pathogen comparedwith a non-pathogenic strain of bacteria or virus.

The invention offers several advantages. Vaccinations performed inaccordance with the invention circumvent the need to identify specifictumor rejection antigens or pathogen antigens, because the correctantigen(s) is automatically selected from the tumor- or pathogen-derivedRNA. If desired, the risk of generating an autoimmune response can bediminished by using tumor-specific RNA. In addition, vaccination withcells loaded with unfractionated tumor-derived RNA likely elicits immuneresponses to several tumor antigens, reducing the likelihood of “escapemutants.” The invention also extends the use of active immunotherapy totreating cancers for which specific tumor antigens have not yet beenidentified, which is the vast majority of cancers. The invention can beused efficaciously even if the tumor itself displays poorimmunogenicity. In addition, the invention is useful for reducing thesize of preexisting tumors, including metastases even after removal ofthe primary tumor. Finally, the invention offers the advantage thatantigen-presenting cells that are loaded with in vitro transcribed RNAcan be more potent vaccines than are antigen-presenting cells that areloaded with peptide antigens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating primary OVA-specific CTL induction invitro with dendritic cells pulsed with RNA. DC were pulsed with totalRNA or poly A⁺ RNA obtained from E.G7-OVA or 'EL4 cells, or in vitrotranscribed OVA RNA in the presence of the cationic lipid DOTAP asdescribed herein. DC pulsed with the OVA peptide were used forcomparison. DC and naive T cells were incubated for 5 days at a R/S of20:1. Viable lymphocytes were harvested, and the CTL activity wasdetermined in a routine europium release assay. E.G7-OVA and EL4 cellswere used as targets. This experiment was repeated three times withsimilar results.

FIG. 2 is a graph illustrating that the sensitization of E.G7-OVA RNApulsed DC for stimulation of OVA-specific primary CTL responses ismediated by the poly A⁺ fraction of RNA. DC were pulsed with total RNA,poly A⁻ RNA or poly A⁺ RNA, and cultured with naive T cells in 96-wellU-bottom plates for 5 days. The poly A⁺ RNA fraction from E.G7-OVA cellswas treated with an antisense oligonucleotide specific for the CTLepitope encoding region of the OVA gene, or a control oligonucleotidefollowed by RNase H treatment to eliminate the hybridized RNA. DC pulsedwith OVA peptide was used as a control. E.G7-OVA, EL4, and RMA cellspulsed with the OVA peptide were used as targets.

FIG. 3 is a histogram depicting the induction of anti-tumor immunity invivo in mice following a single immunization with DC pulsed with RNA. DCwere pulsed with either total or poly A⁺ RNA from E.G7-OVA cells or EL4cells, or with in vitro transcribed OVA RNA or control antisense OVARNA. Mice were immunized with 2×10⁶ DC or 5×10⁶ irradiated E.G7-OVA orEL4 cells injected intraperitoneally, followed by a challenge with 2×10⁷live E.G7-OVA cells. Mice were periodically examined for tumor growth,and were sacrificed when the tumor diameter reached 3-4 cm. All micewere sacrificed at 35-40 days post-challenge.

FIG. 4 is a histogram depicting the regression of spontaneous metastasisin mice vaccinated with DC pulsed with poly A⁺ RNA or total RNA in theB16-F10.9 melanoma model. Mice received by intrafootpad injection liveF10.9 cells, and the legs were amputated when the tumor diameter reached5.5-7.5 mm. Vaccinations were initiated 2 days post-amputation, and werefollowed by two more vaccinations at weekly intervals. Mice werevaccinated intraperitoneally with 2×10⁶ total, poly A⁻ or poly A⁺ RNApulsed DC, or irradiated F10.9 cells or F10.9/K1 cells, or PBS (as acontrol.) Mice were sacrificed based on the metastatic death in thenon-immunized or control groups (28-32 days post-amputation). Metastaticloads were assayed by weighing the lungs and by counting the number ofmetastatic nodules.

DETAILED DESCRIPTION

Before providing detailed working examples of the invention, certainparameters of the invention will be described generally.

A variety of methods are suitable for producing the tumor- orpathogen-derived RNA that can be used in the invention. As the followingexamples illustrate, it is not necessary that the RNA be provided to theAPC in a purified form. Preferably, the RNA sample (i.e., thefractionated tumor preparation or IVT RNA sample) is at least 50%, morepreferably 75%, 90%, or even 99% RNA (wt/vol). In practicing theinvention, antigen-presenting cells, preferably professional APC such asdendritic cells and macrophage, are used. Such cells can be isolatedaccording to previously-described procedures.

Any of a variety of methods can be used to produce RNA-containing tumorpreparations. For example, the tumor preparations can be produced bysonicating tumor cells in a mammalian cell culture medium such asOpti-MEM or a buffer such as phosphate buffered saline. Similarly,pathogen-derived RNA can be produced by sonicating pathogenic bacteriaor cells containing a pathogenic virus. Other methods for disruptingcells also are suitable, provided that the method does not completelydegrade the tumor- or pathogen-derived RNA. Typically, the RNApreparation has 10⁶ to 10⁸ cells/ml; most preferably 10⁷ cells/ml. Asalternatives, or in addition, to sonication, the tumor- orpathogen-derived RNA can be prepared by employing conventional RNApurification methods such as guanidinium isothiocyanate methods and/oroligo dT chromatography methods for isolating poly A⁺ RNA. IVT RNA,synthesized according to conventional methods, can be used in lieu ofRNA in tumor preparations. For example, RNA from a tumor or pathogen canbe reverse transcribed into cDNA, which then is amplified byconventional PCR techniques to provide an essentially unlimited supplyof cDNA corresponding to the tumor or pathogen RNA antigen. Conventionalin vitro transcription techniques and bacterial polymerases then areused to produce the IVT RNA. As an alternative, the IVT RNA can besynthesized from a cloned DNA sequence encoding a tumor or pathogenpolypeptide antigen. Methods for identifying such antigens are known inthe art; for example, several melanoma peptide antigens have beenidentified. RNA transcribed in vitro from cDNA encoding identifiedpeptide antigens can serve as tumor- or pathogen-specific RNA in theinvention. As an alternative, RNA can be transcribed from “minigenes”consisting of a portion of the tumor antigen cDNA that encodes anepitope. Tumor- or pathogen-specific RNA can also be produced byemploying conventional techniques for subtractive hybridization. Forexample, an RNA sample from tumor cells and non-tumor cells can be usedin the subtractive hybridization method to obtain tumor-specific RNA.

Art-known transfection methods are suitable for introducing the tumor-or pathogen-derived RNA into an antigen-presenting cell. For example,5-50 μg of RNA in 500 μl of Opti-MEM can be mixed with a cationic lipidat a concentration of 10 to 100 μg, and incubated at room temperaturefor 20 to 30 minutes. Other suitable lipids include LIPOFECTIN™ (1:1(w/w) DOTMA:DOPE), LIPOFECTAMINE™ (3:1 (w/w) DOSPA:DOPE), DODAC:DOPE(1:1), CHOL:DOPE (1:1), DMEDA, CHOL, DDAB, DMEDA, DODAC, DOPE, DORI,DORIE, DOSPA, DOTAP, and DOTMA. The resulting RNA-lipid complex is thenadded to 1-3×10⁶ cells, preferably 2×10⁶, antigen-presenting cells in atotal volume of approximately 2 ml (e.g., in Opti-MEM), and incubated at37° C. for 2 to 4 hours. Alternatively, the RNA can be introduced intothe antigen presenting cells by employing conventional techniques, suchas electroporation or calcium phosphate transfection with 1-5×10⁶ cellsand 5 to 50 μg of RNA. Typically, 5-20 μg of poly A⁺ RNA or 25-50 μg oftotal RNA is used.

When the RNA is provided as a tumor or pathogen preparation, thepreparation typically is fractionated or otherwise treated to decreasethe concentration of proteins, lipids, and/or DNA in the preparation,and enrich the preparation for RNA. For example, art-known RNApurification methods can be used to at least partially purify the RNAfrom the tumor cell or pathogen. It is also acceptable to treat the RNApreparation with proteases or RNase-free DNases.

The RNA-loaded antigen-presenting cells of the invention can be used tostimulate CTL proliferation in vivo or ex vivo. The ability of theRNA-loaded antigen-presenting cells to stimulate a CTL response can bemeasured by assaying the ability of the effector cells to lyse targetcells. For example, the commonly-used europium release assay can beused. Typically, 5-10×10⁶ target cells are labeled with europiumdiethylenetriamine pentaacetate for 20 minutes at 4° C. After severalwashes 10⁴ europium-labeled target cells and serial dilutions ofeffector cells at an effector:target ratio ranging from 50:1 to 6.25:1are incubated in 200 μl RPMI 1640 with 10% heat-inactivated fetal calfserum in 96-well plates. The plates are centrifuged at 500×g for 3minutes and the incubated at 37° C. in 5% CO₂ for 4 hours. A 50 μlaliquot of the supernatant is collected, and europium release ismeasured by time resolved fluorescence (Volgmann et al., J. Immunol.Methods 119:45-51, 1989).

EXAMPLES

The following working examples are meant to illustrate, not limit, theinvention. First, the methods used in these examples are described.

Mice

Seven to eight weeks old and retired breeder female C57BL/6 mice(H-2^(b)) were obtained from the Jackson Laboratory (Bar Harbor, Me.).

Cell lines

The F10.9 clone of the B16 melanoma of C57BL/6 origin is a highlymetastatic, poorly immunogenic, and low class I expressing cell line.F10.9/K1 is a poorly metastatic and highly immunogenic cell line derivedby transfecting F10.9 cells with class I molecule, H-2K^(b) cDNA. RMAand RMA-S cells are derived from the Rauscher leukemia virus-induced Tcell lymphoma RBL-5 of C57BL/6 (H-2^(b)) origin. Other cell lines usedwere EL4 (C57BL/6, H-2^(b), thymoma), E.G7-OVA (EL4 cells transfectedwith the cDNA of chicken ovalbumin (OVA), A20(H-2^(d) B cell lymphoma)and L929 (H-2^(k) fibroblasts). Cells were maintained in DMEMsupplemented with 10% fetal calf serum (FCS), 25 mM Hepes, 2 mML-glutamine and 1 mM sodium pyruvate. E.G7-OVA cells were maintained inmedium supplemented with 400 μg/ml G418 (GIBCO, Grand Island, N.Y.) andF10.9/K1 cells were maintained in medium containing 800 μg/ml G418.

Antigen presenting cells and responder T cells

Splenocytes obtained from naive C57BL/6 female retired breeders weretreated with ammonium chloride Tris buffer for 3 minutes at 37° C. todeplete red blood cells. Splenocytes (3 ml) at 2×10⁷ cells/ml werelayered over a 2 ml metrizamide gradient column (Nycomed Pharma AS,Oslo, Norway; analytical grade, 14.5 g added to 100 ml PBS, pH 7.0) andcentrifuged at 600 g for 10 minutes. The dendritic cell-enrichedfraction from the interface was further enriched by adherence for 90minutes. Adherent cells (mostly dendritic cells (DC) and a fewcontaminating macrophage (M) were retrieved by gentle scraping, andsubjected to a second round of adherence at 37° C. for 90 minutes todeplete the contaminating M. Non-adherent cells were pooled as splenicDC and FACS analysis showed approximately 80%-85% DC (mAb 33D1), 1-2% M(mAb F4/80), 10% T cells, and <5% B Cells (data not shown).

The pellet was resuspended and enriched for M by two rounds ofadherence at 37° C. for 90 minutes each. More than 80% of the adherentpopulation was identified as M by FACS analysis, with 5% lymphocytesand <55% DC.

B cells were separated from the non-adherent population (B and T cells)by panning on anti-Ig coated plates. The separated cell population,which was comprised of >80% T lymphocytes by FACS analysis was used asresponder T cells.

Isolation of total and poly A⁺ cellular RNA

Total RNA was isolated from actively growing tissue culture cells aspreviously described (Chomczynski and Sacchi, 1987, Analy. Biochem. 162:156-159). Briefly, 10⁷ cells were lysed in 1 ml of guanidiniumisothiocyanate (GT) buffer (4 M guanidinium isothiocyanate, 25 mM sodiumcitrate, pH 7.0; 0.5% sarcosyl, 20 mM EDTA, and 0.1 M2-mercaptoethanol). Samples were vortexed, and followed by sequentialaddition of 100 μl 3 M sodium acetate, 1 ml water-saturated phenol and200 μl chloroform:isoamyl alcohol (49:1). Suspensions were vortexed andthen placed on ice for 15 minutes. The tubes were centrifuged at10000×g, at 4° C. for 20 minutes, and the supernatant was carefullytransferred to a fresh tube. An equal volume of isopropanol was addedand the samples were placed at −20° C. for at least 1 hour. RNA waspelleted by centrifugation as above. The pellet was resuspended in 300μl GT buffer, and then transferred to a microcentrifuge tube. RNA wasagain precipitated by adding an equal volume of isopropanol and placingthe tube at −20° C. for at least 1 hour. Tubes were microcentrifuged athigh speed at 4° C. for 20 minutes. Supernatants were decanted, and thepellets were washed once with 70% ethanol. The pellets were allowed todry at room temperature and then resuspended in TE (10 mM Tris-HCl, 1 mMEDTA, pH 7.4). Possible contaminating DNA was removed by incubating theRNA sample in 10 mM MgCl₂, 1 mM DTT and 5 U/ml RNase-free DNase(Boehringer-Mannheim) for 15 minutes at 37° C. The solution was adjustedto 10 mM Tris, 10 mM EDTA, 0.5% SDS and 1 mg/ml Pronase(Boehringer-Mannheim), followed by incubation at 37° C. for 30 minutes.Samples were extracted once with phenol-chloroform and once withchloroform; RNA was again precipitated in isopropanol at −20° C.Following centrifugation, the pellets were washed with 70% ethanol, thenair dried and resuspended in sterile water. Total RNA was quantitated bymeasuring the optical density (OD) at 260 and 280 nm. The OD 260/280ratios were typically 1.65-2.0. The RNA was stored at −70° C.

Poly A⁺ RNA was isolated either from total RNA using an OLIGOTEX™ polyA⁺ purification kit (Qiagen), or directly from tissue culture cellsusing the Messenger RNA Isolation kit (Stratagene) as per themanufacturer's protocols. If desired, alternative, conventional methodscan be used to prepare poly A⁺ RNA.

Production of in vitro transcribed RNA

The 1.9 kb EcoRI fragment of chicken ovalbumin cDNA in pUC18 (McReynoldset al., 1978, Nature 273:723) containing the coding region and 3′untranslated region, was cloned into the EcoRI site of pGEM4Z (Promega).Clones containing the insert in both the sense and anti-senseorientations were isolated, and large scale plasmid preps were madeusing Maxi Prep Kits TM plasmid preparation kit (Qiagen). Plasmids werelinearized with BamHI for use as templates for in vitro transcription.Transcription was carried out at 37° C. for 3-4 hours using theMEGAscript In Vitro Transcription Kit TM (Ambion) according to themanufacturer's protocol and adjusting the GTP concentration to 1.5 mMand including 6 mM m⁷G(5¹)ppp(5¹)G cap analog (Ambion). Other,conventional in vitro transcription methods also are suitable. TemplateDNA was digested with RNase-free DNase 1, and RNA was recovered byphenol:chloroform and chloroform extraction, followed by isopropanolprecipitation. RNA was pelleted by microcentrifugation, and the pelletwas washed once with 70% ethanol. The pellet was air-dried andresuspended in sterile water.

RNA was incubated for 30 minutes at 30° C. in 20 mM Tris-HCl, pH 7.0, 50mM KCl, 0.7 mM MnCl₂, 0.2 mM EDTA, 100 μg/ml acetylated BSA, 10%glycerol, 1 mM ATP and 5000 U/ml yeast poly (A) polymerase (UnitedStates Biochemical). The capped, polyadenylated RNA was recovered byphenol:chloroform and chloroform extraction followed by isopropanolprecipitation. RNA was pelleted by microcentrifugation, and the pelletwas washed once with 70% ethanol. The pellet was air-dried andresuspended in sterile water. RNA was quantitated by measuring the OD at260 and 280 nm, and the RNA stored at −70° C.

Oligodeoxynucleotide directed cleavage of OVA mRNA by RNase H

The procedure used for RNase H site-specific cleavage of ovalbumin mRNAwas adapted from those previously described (Donis-Keller, 1979, Nucl.Acid. Res. 7: 179-192). Briefly, 5-10 μg mRNA from E.G7-OVA cells wassuspended in 20 mM HEPES-KOH, pH 8.0, 50 mM KCl, 4 mM MgCL₂, 1 mM DTT,50 μg/ml BSA and 2 μM of either the oligodeoxynucleotide 5′ CAG TTT TTCAAA GTT GAT TAT ACT-3′SEQ ID NO:1, which hybridizes to sequence in OVAmRNA that codes for the CTL epitope SIINFEKL, or 5′-TCA TAT TAG TTG AAACTT TTT GAC-3′(SEQ ID NO:2) (Oligos, Etc.), which serves as a negativecontrol. The samples were heated to 50° C. for 3 minutes followed byincubation at 37° C. for 30 minutes. RNase H (Boehringer-Mannheim) wasadded at 10 U/ml, and digestion proceeded for 30 minutes at 37° C. RNAwas recovered by phenol:choloroform and chloroform extraction, followedby isopropanol precipitation. RNA was pelleted by microcentrifugation,and the pellet was washed once with 70% ethanol. The pellet then wasair-dried and resuspended in sterile water. Cleavage of OVA mRNA wasconfirmed by oligo dT primed reverse transcription of test and controlsamples, followed by PCR with OVA specific primers that flank thecleavage site. PCR with actin-specific primers was used to controlbetween test and control samples.

Pulsing of APC

APC were washed twice in Opti-MEM medium (GIBCO, Grand Island, N.Y.).Cells were resuspended in Opti-MEM medium at 2-5×10⁶ cells/ml, and addedto 15 ml polypropylene tubes (Falcon). The cationic lipid DOTAP(Boehringer Mannheim Biochemicals, Indianapolis, Ind.) was used todeliver RNA into cells (Walker et al., 1992, Proc. Natl. Acad. Sci. USA89: 7915-7918). RNA (in 250-500 μl Opti-MEM medium) and DOTAP (in250-500 μl Opti-MEM medium) was mixed in a 12×75 mm polystyrene tube atroom temperature (RT) for 20 minutes. The RNA to DOTAP ratio routinelyused was 1:2, and varied in certain experiments between 2:1 to 1:2. Thecomplex was added to the APC (2-5×10⁶ cells) in a total volume of 2 mland incubated at 37° C. in a water-bath with occasional agitation for 2hours. The cells were washed and used as stimulators for primary CTLinduction in vitro.

The synthetic peptide encoding the CTL epitope in chicken ovalbumin OVA,aa 257-264 SIINFEKL (H-2K^(b)), was used for peptide pulsing. Thepeptide had unblocked (free) amino and carboxyl ends (Research Genetics,Birmingham, Ala.). Peptides were dissolved in serum-free IMDM and storedat −20° C.

Induction of CTL in vitro

T cells (5×10⁶ cells/ml) and RNA or peptide pulsed APC (2.5×10⁵cells/ml) were cultured in IMDM with 10% FCS, 1 mM sodium pyruvate, 100IU/ml penicillin, 100 mg/ml streptomycin, and 5×10⁻⁵ M β-mercaptoethanolin 96 well U-bottom plates to give an R/S ratio of 20:1. After 5 days,cells were used as effectors in a standard 4 hours europium releaseassay.

Cytotoxicity assay

In these assays, 5-10×10⁶ target cells were labeled with europiumdiethylenetriamine pentaacetate for 20 minutes at 4°. After severalwashes, 10⁴ europium-labeled targets and serial dilutions of effectorcells at effector:target ratios of 50:1 to 6.25:1 were incubated in 200μl of RPMI 1640 with 10% heat-inactivated FCS in 96-well V-bottomplates. The plates were centrifuged at 500 g for 3 minutes and incubatedat 37° C. and 5% CO₂ for 4 hours. 50 μl of the supernatant washarvested, and europium release was measured by time resolvedfluorescence (Delta fluorometer, Wallace Inc., Gaithersburg, Md.).Spontaneous release was less than 25%. Standard errors (SE) of the meansof triplicate cultures was less than 5%.

Immunotherapy

E.G7-OVA model

C57BL/6 mice were immunized once with irradiated, RNA-pulsed APC (2×10⁶cells/mouse) or 5×10⁶ E.G7-OVA or EL4 cells. At 10-14 dayspost-immunization, mice were challenged with 2×10⁷ live E.G7-OVA cellsinjected sub-cutaneously in the flank region. Mice were monitored on aregular basis for tumor growth and size. Mice with tumor sizes >3.5 cmwere sacrificed. All survivors were sacrificed at 40 dayspost-challenge.

F10.9-B16 melanoma model

Mice were received by intrafootpad injection 2×10⁵ F10.9 cells. Thepost-surgical protocol was essentially as described previously (Porgadoret al., 1995, Cancer Res. 55: 4941-4949). The legs of the mice wereamputated when the local tumor in the footpad was 5.5-7.5 mm indiameter. Post-amputation mortality was less than 5%. At two dayspost-amputation, the mice were immunized intraperitoneally, followed byweekly vaccinations twice, for a total of three vaccinations. The micewere sacrificed based on the metastatic death in the non-immunized orcontrol groups (at 28-32 days post-amputation). The metastatic loadswere assayed by weighing the lungs and by counting the number ofmetastatic nodules.

Induction of a primary CTL response in vitro using dendritic cellstransfected with chicken ovalbumin RNA

The ability of RNA pulsed splenic dendritic cells (DC) derived fromC57BL/6 (H-2K^(b)) mice to induce a primary CTL response in vitro wasdemonstrated in the E.G7-OVA tumor system. E.G7-OVA cells were derivedfrom the EL4 tumor cell line (H-2K^(b) haplotype) by transfection withthe chicken ovalbumin cDNA (Moore et al., 1988, Cell 54: 777-785). Thechicken ovalbumin encodes a single dominant epitope (aa 257-264) inC57BL/6 mice (Rotzschke et al., 1991, Euro. Journal Immunology, 21:2891-2891).

Dendritic cells pulsed with the OVA peptide (aa 257-264) incubated withT cells from naive mice induce a potent CTL response in vitro (FIG. 1).This example demonstrates that RNA can be used as a source of antigen tosensitize DC to present antigen to CD8⁺ T cells. Splenic DC wereisolated from C57BL/6 mice and pulsed with OVA peptide or incubated withRNA synthesized in vitro (OVA IVT RNA) from a plasmid encoding thechicken ovalbumin cDNA, and used to stimulate an OVA-specific primaryCTL response in vitro. As shown in FIG. 1, both OVA peptide as well asOVA IVT RNA pulsed DC were capable of inducing an OVA specific primaryCTL response (FIG. 1). RNA pulsed DC were consistently more effectivestimulators than peptide pulsed DC. To test whether RNA isolated fromE.G7-OVA cells was capable of sensitizing DC to stimulate a primary,OVA-specific, CTL response, total RNA or poly A⁺ RNA was isolated fromE.G7-OVA or EL4 cells and incubated with DC. As shown in FIG. 1, DCpulsed with either total or poly A⁺ RNA from E.G7-OVA cells but not fromEL4 cells, were capable of inducing a strong OVA specific CTL response.Surprisingly, DC pulsed with unfractionated RNA, total or poly A⁺, wereas potent inducers of a primary CTL response as DC pulsed with the OVApeptide encoding a defined CTL epitope. Stimulation of a CTL response by(total or poly A⁺) EL4 RNA pulsed DC was only marginally abovebackground and statistically not significant (Compare to lysis of EL4targets by CTL stimulated with OVA peptide or OVA IVT RNA pulsed DC),reflecting the immunodominance of the OVA epitope and the relativeweakness of the EL4 encoded antigens.

As is illustrated by FIG. 2, total, as well as poly A⁺, but not poly A⁻,RNA isolated from E.G7-OVA cells is capable of sensitizing DC tostimulate a primary CTL response. To prove that sensitization of DC isindeed mediated by RNA, poly A⁺ RNA from E.G7-OVA cells was incubatedwith either an antisense oligonucleotide spanning the sequence encodingthe single CTL epitope present in the chicken ovalbumin gene or with acontrol oligodeoxynucleotide, and then treated with RNase H to removeany RNA sequence to which the oligodeoxynucleotide probe has hybridized.As shown in FIG. 2, induction of a primary, OVA-specific CTL responsewas abolished when the poly A⁺ RNA was incubated with the antisense, butnot with the control, oligodeoxynucleotide. FIG. 2 also shows that cellsexpressing the complete ovalbumin gene, E.G7-OVA cells, and RMA-S cellspulsed with the 8 amino acid long OVA peptide encoding the singledominant CTL epitope are lysed to a similar extent following stimulationwith total or poly A⁺ E.G7-OVA RNA pulsed DC. This indicates, therefore,that the majority of epitopes presented by E.G7-OVA RNA pulsed DCcorrespond to the previously defined single dominant CTL epitope encodedin the chicken ovalbumin gene.

Induction of anti-tumor immunity by DC pulsed with tumor RNA

This example demonstrates that vaccination of mice with OVA RNA pulsedDC provided protection against a challenge with E.G7-OVA tumor cells.Mice were immunized once with 2×10⁶ RNA pulsed DC or with 5×10⁶irradiated E.G7-OVA cells. Ten days later, mice were challenged with atumorigenic dose of E.G7-OVA cells. Appearance and size of the tumorwere determined on a regular basis. FIG. 3 shows the size of the tumorsat 37 days post-tumor implantation. The average tumor size in miceimmunized with irradiated EL4 cells was 25 cm, while the average tumorsize in animals immunized with the OVA expressing EL4 cells (E.G7-OVA)was only 7.03 cm. This difference is a reflection of the highimmunogenicity of the chicken OVA antigen expressed in EL4 cells and thepoor immunogenicity of the parental, EL4, tumor cell line. Vaccinationwith DC pulsed with RNA (total or poly A⁺ fraction) derived fromE.G7-OVA cells was as effective as vaccination with the highlyimmunogenic E.G7-OVA cells (average tumor size 7 cm). Vaccination withDC incubated with total or poly A⁺ RNA derived from EL4 tumor cells hada slight protective effect (average tumor size: 22 cm and 19.5 cm,respectively) which was not statistically significant, consistent withpoor to undetectable immunogenicity of EL4-derived antigens. Consistentwith the primary CTL induction data (FIG. 1), vaccination of mice withOVA IVT RNA pulsed DC provided the most effective antitumor response(average tumor size: 3.9 cm), while vaccination with the controlantisense OVA IVT RNA did not elicit a significant protective response.

The potency of DC pulsed with tumor-derived RNA was further evaluated inthe B16/F10.9 (H-2^(b)) melanoma metastasis model. The B16/F10.9melanoma tumor is poorly immunogenic, expresses low levels of MHC classI molecules, and is highly metastatic in both experimental andspontaneous metastasis assay systems (Porgador et al., 1996, J.Immunology 156: 1772-1780). Porgador et al. have shown that, whenvaccinations are carried out after the removal of the primary tumorimplant, only irradiated tumor cells transduced with both the IL-2 andthe H-2K^(b) genes, are capable of significantly impacting themetastatic spread of B16/F10.9 tumor cells in the lung (Porgador et al.1995, Cancer Research 55: 4941-4949). Thus, the B16/F10.9 melanoma modeland the experimental design used by Porgador et al. constitutes astringent and clinically relevant experimental system to assess theefficacy of adjuvant treatments for metastatic cancer.

To demonstrate that immunization with tumor RNA pulsed DC, in accordancewith the invention, was capable of causing the regression of preexistinglung metastases, primary tumors were induced by implantation ofB16/F10.9 tumor cells in the footpad. When the footpad reached 5.5-7.5mm in diameter, the tumors were surgically removed. Two days later, micewere immunized with irradiated B16/F10.9 cells, irradiated B16/F10.9cells transduced with the H-2K^(b) gene (F10.9K1), or with RNA pulsed DCpreparations (FIG. 4). The mice received a total of three vaccinationsgiven at weekly intervals. The average lung weight of a normal mouse is0.18-0.22 g. Mice treated with PBS (a negative control) were overwhelmedwith metastases. The mean lung weight of mice in this treatment groupwas 0.81 g; approximately three-quarters of the weight was contributedby the metastases, which were too many to count (>100 nodules). Asimilar metastatic load was seen when mice were treated with irradiatedB16/10.9 cells (data not shown), which confirms numerous previousobservations that treatment with irradiated B16/F10.9 tumor cells alonehas no therapeutic benefit in this tumor model. As also previouslyshown, immunization with H-2K^(b) expressing B16/F10.9 cells (F10.9K1,as a positive control) had a modest therapeutic benefit, as indicated bya statistically significant decrease in the average lung weight of theanimals in this treatment group. A dramatic response, however, was seenin animals treated with DC that were pulsed with total RNA derived fromF10.9 cells in accordance with the invention. The mean lung weight ofmice in this treatment group was 0.37 g. A significant dramatic responsealso was seen in mice treated with DC pulsed with poly A⁺ RNA derivedfrom F10.9 cells in accordance with the invention (average lung weight:0.42 g). By contrast, no statistically significant decrease inmetastatic load was seen in mice treated with DC that were pulsed witheither the poly A- RNA fraction derived from F10.9 cells or with totalRNA isolated from EL4 tumor cells.

The observation that cells expressing the OVA protein (E.G7-OVA) orcells pulsed with the OVA peptide were efficiently lysed by CTL, and thesensitization of DC fractionated with poly A⁺ RNA, strongly suggest thatRNA-mediated stimulation of CTL occurs via translation of the input RNAand generation of the predicted class I restricted epitopes, in thiscase a single dominant epitope encoded in the chicken OVA peptide. Thesedata show that RNA mediated sensitization of DC is more effective thanpulsing with peptide because the transfected RNA can serve as acontinuous source for the production of antigenic peptides.

Therapeutic Use

The invention can be used to treat or prevent tumor formation in apatient (e.g., melanoma tumors, bladder tumors, breast cancer tumors,colon cancer tumors, prostate cancer tumors, and ovarian cancer tumors).Similarly, the invention can be used to treat or prevent infection in apatient with a pathogen such as a bacterium (e.g., Salmonella, Shigella,or Enterobacter) or a virus (e.g., a human immunodeficiency virus, aHerpes virus, an influenza virus, a poliomyelitis virus, a measlesvirus, a mumps virus, or a rubella virus).

In treating or preventing tumor formation or pathogen infection in apatient, it is not required that the cell(s) that is administered to thepatient be derived from that patient. Thus, the antigen-presenting cellcan be obtained from a matched donor, or from a culture of cells grownin vitro. Methods for matching haplotypes are known in the art.Similarly, it is not required that the RNA be derived from the patientto be treated. RNA from a donor can be used.

It is preferable that treatment begin before or at the onset of tumorformation or infection, and continue until the cancer or infection isameliorated. However, as the examples described herein illustrate, theinvention is suitable for use even after a tumor has formed, as theinvention can cause a regression of the tumor. In treating a patientwith a cell or vaccine produced according to the invention, the optimaldosage of the vaccine or cells depends on factors such as the weight ofthe mammal, the severity of the cancer or infection, and the strength ofthe CTL epitope. Generally, a dosage of 10⁵ to 10⁸ RNA-loadedantigen-presenting cells/kg body weight, preferably 10⁶ to cells/kg bodyweight, should be administered in a pharmaceutically acceptableexcipient to the patient. The cells can be administered by usinginfusion techniques that are commonly used in cancer therapy (see, e.g.,Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimaldosage and treatment regime for a particular patient can readily bedetermined by one skilled in the art of medicine by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

Where the antigen-presenting cell is used to induce a CTL response invitro, the resulting effector CTLs can subsequently be administered to amammal in a CTL-based method of therapy (see, e.g., PCT/US91/06441). CTLproduced in vitro with the antigen-presenting cells of the invention canbe administered in a pharmaceutically acceptable excipient to a mammalby employing conventional infusion methods (see, e.g., Rosenberg et al.,supra). Typically, 10⁹-10¹⁰ cells are administered over the course of 30minutes, with treatment repeated as necessary. Such a CTL-based methodof therapy may be combined with other methods, such as directadministration of the antigen-presenting cells of the invention. The CTLand antigen-presenting cells may be autologous or heterologous to thepatient undergoing therapy. If desired, the treatment may also includeadministration of mitogens (e.g., phyto-hemagglutinin) or lymphokines(e.g., IL-2 or IL-4) to enhance CTL proliferation.

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What is claimed is:
 1. A method for producing an RNA-loaded antigenpresenting cell (APC), said method comprising: introducing into anantigen-presenting cell in vitro RNA selected from the group consistingof (i) RNA comprising tumor-specific RNA and (ii) RNA comprisingpathogen-specific RNA, thereby producing an RNA-loaded APC.
 2. Themethod of claim 1, wherein said APC is a dendritic cell.
 3. The methodof claim 1, wherein said APC is a macrophage.
 4. The method of claim 1,wherein said APC is an endothelial cell.
 5. The method of claim 1,wherein said APC is an artificially generated APC.
 6. The method ofclaim 1, wherein said RNA comprises tumor specific RNA that comprisespoly A⁺ RNA.
 7. The method of claim 1, wherein said RNA comprisestumor-specific RNA that comprises cytoplasmic RNA.
 8. The method ofclaim 1, wherein said RNA corresponds to a tumor antigen.
 9. The methodof claim 1, wherein said RNA corresponds to a pathogen antigen.
 10. Themethod of claim 1, wherein said RNA corresponds to an epitope.
 11. Themethod of claim 1, wherein said RNA is tumor-specific RNA.
 12. Themethod of claim 1, wherein the RNA is introduced into the APC bycontacting the APC with the RNA in the presence of a cationic lipid. 13.The method of claim 1, wherein said RNA comprises tumor-specific RNAthat is provided as a tumor extract that is fractionated with respect toa non-RNA component of the tumor.
 14. An RNA-loaded APC produced by themethod of claim
 1. 15. The method of claim 1, wherein the tumor-specificRNA comprises melanoma-specific RNA.
 16. The method of claim 1, whereinthe tumor-specific RNA comprises bladder tumor-specific RNA.
 17. Themethod of claim 1, wherein the tumor-specific RNA comprisestumor-specific RNA selected from the group consisting of breastcancer-specific RNA, colon cancer-specific RNA, prostate cancer-specificRNA and ovarian cancer-specific RNA.
 18. The method of claim 1, whereinsaid pathogen-specific RNA comprises viral RNA.
 19. The method of claim18, wherein said virus is selected from the group consisting ofHepatitis viruses, human immunodeficiency viruses, influenza viruses,poliomyelitis viruses, measles viruses, herpes viruses, mumps viruses,and rubella viruses.
 20. The method of claim 1, wherein saidpathogen-specific RNA comprises bacterial RNA.
 21. The method of claim20, wherein said bacterium is selected from the group consisting ofSalmonella, Shigella, and Enterobacter.
 22. The method of claim 1,wherein said RNA is isolated from a cell.
 23. The method of claim 1,wherein said RNA is prepared by PCR amplification and in vitrotranscription.
 24. The method of claim 1, wherein said RNA istumor-specific RNA that comprises nuclear RNA.
 25. The method of claim 1wherein said RNA corresponds to a minigene.
 26. The method according toclaim 1 wherein said RNA consists of tumor-specific RNA encoding aspecific antigen.
 27. The method according to claim 1 wherein said RNAconsists of pathogen-specific RNA encoding a specific pathogen antigen.28. The method according to claim 1 wherein said APC is a professionalAPC.
 29. The method according to claim 1 wherein said RNA is transcribedfrom cloned cDNA.